Early search for new promoters in S. pombe yielded adh1, a strong but constitutive promoter, which drives the expression of the glycolytic enzyme alcohol dehydrogenase. This promoter has been used in the vectors pART1 and pEVP11 (Russell, 1989, In: Molecular Biology of the Fission Yeast. San Diego: Academic Press, Inc. Nasim. A., Young, P., and Johnson, B. F., Eds. pp. 243-71). Among the inducible promoters are the glucose regulated promoter fructose bisphosphase (fbp) (Hoffman and Winston, 1989, Gene 84: 473-79) and the invertase promoter (inyl) (Tanaka et al., 1998, Biochem. Biophys. Res. Commun. 245: 246-53), which have been used successfully to express a number of proteins including GFP. The strongest known promoter in S. pombe is adh1, which is a constitutively expressed promoter. Although expression driven by this promoter can yield levels of protein to the extent of 0.5-2% of total cellular protein (Russell, 1989, In Molecular Biology of the Fission Yeast. San Diego: Academic Press, Inc. Nasim. A., Young, P., and Johnson, B. F., Eds. pp. 243-271), it can pose difficulties when the expression of toxic proteins is desired. Therefore, researchers have always focused their attention to the development of inducible promoters.
The regulatable promoters in S. pombe include the fbp, inv1 and nmt1. The fbp1 promoter is repressed by 8% glucose and derepressed in 0.1% glucose plus 3% maltose (Hoffman and Winston, 1989, Gene 84: 473-479). A limitation of this promoter is that it is derepressed in stationary phase and furthermore, the cells expressing the vector do not grow well under the inducing conditions. A similar drawback is faced by the inv1 promoter derived from the inv1gene that codes for invertase in S. pombe (Tanaka et al., 1998, Biochem. Biophys. Res. Commun. 245: 246-53). It is also repressed by glucose and derepressed by depletion of glucose. The regulation of expression using this promoter cannot be very tight because of progressive depletion of glucose in the culture medium as a result of its utilization during the cellular growth. Likewise another inducible promoter of S. pombe be acid phosphatase structural gene (pho1), which is induced by low concentrations of inorganic phosphate in the medium, has the drawback that it shows a significant level of uninduced transcription, thus negating its potential use as a promoter (Maundrell, 1990, J. Biol. Chem. 265: 10857-64). Thus, to date only one promoter element has come to be used regularly as a research tool, namely nmt1, which is repressed by high concentration of thiamin and induced by absence of thiamin (Maundrell, 1990, J. Biol. Chem. 265: 10857-64). The most common and the strongest form of this promoter is nmt1. A few derivatives of the nmt1 promoter were subsequently developed that yield very high (nmt1), medium (nmt41) and low (nmt81) levels of expression (Forsburg, 1993, Nucleic Acid Res. 21: 2955-56). A related problem of these promoters is their leakiness even under repressed conditions and the leakiness appears to be directly proportional to the promoter strength (Forsburg, 1993, Nucleic Acid Res. 21: 2955-2956). The induction regime involves growth of cells harboring the plasmid expressing a particular gene under the control of the nmt1 promoter in presence of thiamin. After growing to early log phase (OD600 of ˜0.3), cells are washed with and transferred to a synthetic medium lacking thiamin and grown further. The expression of the gene of interest is observed after nearly 18-20 hours of growth in the medium lacking thiamin. Apart from the cumbersome problem of handling cells under sterile conditions through the steps of washing and resuspension in thiamin-free medium, the other major problem with this promoter is that it is leaky, that is, the expression of the gene is never completely repressed in the presence of thiamin. This can lead to a deleterious effect on the growth rate of cells even before the start of induction because of possible metabolic load. A similar effect may be exerted during induction because of the long time of induction to achieve full expression level. The presence of the heterologous protein during the long induction period in the intracellular milieu may also lead to cellular defect and protein degradation. The present invention therefore obviates these drawbacks and through the process of this invention a temperature sensitive (or regulated) promoter based vector for expression of heterologous proteins in fission yeast, Schizosaccharomyces pombe has been developed. This invention is particularly useful in efficient, economic and regulated expression of proteins both homologous and heterologous.
The new promoter elements are isolated by screening of promoters which allow expression of a Green fluorescent protein (GFP) reporter gene in response to a shift in temperature. The promoter elements thus isolated represent a truncated region of the previously reported no-message for thiamine 1 (nmt1) promoter (Maundrell, 1990, J. Biol. Chem. 265: 10857-64) having some unique properties that make them more advantageous to use as compared to other known promoters including nmt1 in Schizosaccharomyces pombe (S. pombe ).
These characteristics are: i) temperature sensitive expression: induction of expression by shifting the temperature from 36° C. to 25° C., ii) faster kinetics of expression, iii) moderate level of expression, iv) low leaky expression and v) lack of toxicity.
Selection of a suitable promoter is the most important factor in obtaining optimum level of expression. In early studies, several promoters that work in Saccharromyces cerevisiae (S. cerevisiae) were tried in S. pombe, but with limited success. The promoters that provided a good level of expression included phosphoglycerate kinase (PGK), alcohol dehydrogenase I (ADH1) and iso-1-cytochrome c (CYC). Among other heterologous promoters that work in S. pombe are the simian virus (SV40) promoter, human cytomegalovirus (hCMV) promoter, cauliflower mosaic virus (CaMV) 35S promoter, tomato nitrate reductase promoter, human serum albumin TATA element, adenovirus region 3 promoter, human immunodeficiency virus-1 Long terminal repeat (HIV-1 LTR) promoter etc. Another expression system combines the CaMV promoter and tetracycline regulatory sequences to elicit tetracycline regulated expression. Recently, a co-transformation strategy using a vector containing the hCMV promoter and another vector containing the autonomous replication sequence (ars1) and stable (stb) elements has been reported {review by Giga-Hama, 1997, in Foreign Gene Expression in Fission Yeast Schizosaccharomyces pombe (Giga-Hamma and Kumagai, Eds.), Springer-Verlag, Berlin pp. 3-28}.
The advantages of the new promoters vis-a-vis the nmt1 and its other known derivative promoters strongly support the aspects of novelty and lack of obviousness and anticipation. In fact, literature published on the characteristics of the nmt1 promoters have only focussed on the minimal size of the nmt1 promoters that is repressible by thiamin and on the role of trans-acting factors. No investigation has envisaged or anticipated in published literature whether derivatives of the nmt1 promoter having altogether new characteristics different from the original parent promoter could be derived. In fact, from our screen we were expecting to isolate heat-inducible promoters, like heat shock promoters, rather than the temperature sensitive ones that we actually obtained. Thus, identification of promoter that is heat sensitive, was an unexpected discovery for us as well. Therefore, we believe that these points will strengthen the claims for lack of obviousness and anticipation in obtaining the present set of promoters.